In clinical testing and diagnostics, many reagent strips used to analyze urine contain tests for urobilinogen. Urobilinogen is a reduction product of bilirubin which itself is formed from the degradation of the red blood cells. Normally, there is little or no urobilinogen excreted into the urine unless a pathological condition exists.
The presence of abnormally high levels of urobilinogen in a patient's urine is possibly indicative of hepatic diseases, hemolytic diseases and biliary obstruction. In particular, the determination of abnormally high urobilinogen levels is particularly useful for detecting the early stages of hepatitis. The commercially available Multistix.RTM. reagent strips of Bayer Corporation, Elkhart, Ind., is a typical example of such reagent strips. The reagent test pad condenses urobilinogen with diethylaminobenzaldehyde under acidic conditions to produce the diethylaminobenzylidenyl urobilinogen responsible for the optical signal. As part of normal quality control, the clinician must be assured that the urobilinogen test is working properly by employing a positive control for the test which mimics the urobilinogen material in a patient sample. A specified amount of urobilinogen is thus added to synthetic or fresh urine that is free of endogenous urobilinogen to produce a control solution. This control solution is then used to monitor the accuracy and precision of urine urobilinogen tests.
Traditionally, liquid urobilinogen is synthesized artificially by reducing bilirubin using a catalyst such as sodium amalgam, vanadium or platinum. There have, however, been problems with these traditional methods of synthesis. Sodium amalgam, for instance, is unstable and causes environmental pollution through mercury contamination. Vanadium and platinum are readily oxidizable, expensive and require special equipment when used as catalysts for bilirubin reduction.
Alternately, Raney nickel, copper or iron can be used to reduce bilirubin to urobilinogen. Raney catalysts are powdered metals of nickel, copper, or iron prepared from an alloy of the metal and aluminum by dissolving the aluminum in an alkali solution. Urobilinogen produced by utilizing a Raney catalyst requires lyophilization to ensure extended stability. Specifically, 10% or more of liquid urobilinogen produced by reduction with a Raney catalyst degrades significantly in one day even under refrigeration. Lyophilization is an extra step that adds additional costs and labor to urobilinogen production. Lyophilization also forces the clinician to expend additional time reconstituting the lyophilized powder. Such reconstitution may sacrifice precision quantification of urobilinogen content when the product is reconstituted. Furthermore, the protocol involving Raney catalysts is relatively tedious to perform because the reduction reaction must be performed at 10.degree. C. or lower to effect a reaction.
Given the limitations of the existing methods of producing both lyophilized and liquid urobilinogen and the inherent problems associated with a product which must be stored in lyophilized form, there is a clear need for a method of producing a stable liquid urobilinogen composition which can be produced safely, easily and inexpensively.